U.S. patent application number 12/439370 was filed with the patent office on 2010-04-15 for modulation of mlck-l expression and uses thereof.
Invention is credited to Asrar Malik, Dolly Mehta, Shahid S. Siddiqui.
Application Number | 20100093830 12/439370 |
Document ID | / |
Family ID | 39136919 |
Filed Date | 2010-04-15 |
United States Patent
Application |
20100093830 |
Kind Code |
A1 |
Mehta; Dolly ; et
al. |
April 15, 2010 |
Modulation of MLCK-L Expression and Uses Thereof
Abstract
In various aspects and embodiments the invention provides
methods and reagents for controlling gene expression, and for
treating disorders and diseases. Embodiments provide methods and
reagents specifically for the regulation of MLCK expression and for
the use thereof in treating disorders and diseases. Various
embodiments provide methods and reagents for specifically down
regulating the expression of MLCK-L more efficiently than that of
MLCK-S, and for the use thereof in treating disorders and diseases.
Embodiments provide siNA for the same, particularly siRNAs. Various
of the embodiments are useful for the treatment of inflammatory
disorders and diseases, including, for one example in this regard,
Asthma.
Inventors: |
Mehta; Dolly; (Elmhurst,
IL) ; Siddiqui; Shahid S.; (Wilmette, IL) ;
Malik; Asrar; (Hinsdale, IL) |
Correspondence
Address: |
POLSINELLI SHUGHART PC
700 W. 47TH STREET, SUITE 1000
KANSAS CITY
MO
64112-1802
US
|
Family ID: |
39136919 |
Appl. No.: |
12/439370 |
Filed: |
August 30, 2007 |
PCT Filed: |
August 30, 2007 |
PCT NO: |
PCT/US07/77310 |
371 Date: |
August 27, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60841000 |
Aug 30, 2006 |
|
|
|
Current U.S.
Class: |
514/44A ;
435/375; 536/24.5 |
Current CPC
Class: |
C12Y 207/11018 20130101;
A61P 11/06 20180101; A61P 29/00 20180101; C12N 2310/14 20130101;
C12N 15/1137 20130101 |
Class at
Publication: |
514/44.A ;
536/24.5; 435/375 |
International
Class: |
A61K 31/7105 20060101
A61K031/7105; C07H 21/02 20060101 C07H021/02; C12N 5/00 20060101
C12N005/00; A61P 11/06 20060101 A61P011/06; A61P 29/00 20060101
A61P029/00 |
Claims
1. A double stranded short interfering nucleic acid (siNA) molecule
that directs cleavage of a myosin light chain kinase (MLCK) RNA,
wherein: (a) each strand of said siNA molecule is about 19 to about
25 nucleotides in length; and (b) one strand of said siNA molecule
comprises a region having a nucleotide sequence having sufficient
complementarity to the MLCK RNA for the siNA molecule to direct
cleavage of the MLCK RNA via RNA interference.
2. A double-stranded siNA, according to claim 1, wherein the siNA
is a siRNA.
3. A double-stranded siNA according to claim 1, wherein the siNA
cleaves MLCK-L RNA more efficiently than MLCK-S RNA.
4. A double-stranded siNA, according to claim 3, wherein the siNA
is a siRNA.
5. A double-stranded siNA according to claim 1,wherein the siNA
cleaves MLCK-L RNA at least twice as efficiently as it cleaves
MLCK-S RNA.
6. A double-stranded siNA, according to claim 5, wherein the siNA
is a siRNA.
7. A double-stranded siRNA, according to claim 1, wherein the siNA
specifically cleaves MLCK-L RNA and substantially does not cleave
MLCK-S RNA.
8. A double-stranded siNA, according to claim 7, wherein the siNA
is a siRNA.
9. A double-stranded siRNA, according to claim 7, wherein the siRNA
comprises a region having the sequence of SEQ ID NO:3.
10. A method for modulating MLCK expression in a cell, comprising
introducing into a cell a double stranded siNA that cleaves MLCK
RNA, wherein (a) each strand of said siNA molecule is about 19 to
about 25 nucleotides in length; and (b) one strand of said siNA
molecule comprises a region having a nucleotide sequence having
sufficient complementarity to the MLCK RNA for the siNA molecule to
direct cleavage of the MLCK RNA via RNA interference.
11. A method according to claim 10, wherein the siNA specifically
cleaves MLCK-L RNA and substantially does not cleave MLCK-S
RNA.
12. A method according to claim 11, wherein the siNA is a
siRNA.
13. A method according to claim 12, wherein the siRNA comprises a
region having the sequence of SEQ ID NO:3.
14. A method for modulating tissue inflammation in a patient
suffering therefrom, comprising: administering to a patient
suffering from tissue inflammation a nucleic acid in an amount and
by a route effective to modulate said tissue inflammation, wherein
said nucleic acid comprises a region complementary to an RNA
encoded by an MLCK gene and is effective to direct cleavage
specifically of said RNA by RNA interference.
15. A method according to claim 14, wherein said nucleic acid is a
double stranded siNA wherein (a) each strand of said siNA molecule
is about 19 to about 25 nucleotides in length; and (b) one strand
of said siNA molecule comprises a region having a nucleotide
sequence having sufficient complementarity to the MLCK RNA for the
siNA molecule to direct cleavage of the MLCK RNA via RNA
interference.
16. A method according to claim 15, wherein the siNA specifically
cleaves MLCK-L RNA and substantially does not cleave MLCK-S
RNA.
17. A method according to claim 16, wherein the siNA is a
siRNA.
18. A method according to claim 17, wherein the siRNA comprises a
region having the sequence of SEQ ID NO:3.
19. A method for treating asthma in a patient suffering therefrom,
comprising: administering to a patient suffering from asthma a
nucleic acid in an amount and by a route effective to treat asthma,
wherein said nucleic acid comprises a region complementary to an
RNA encoded by an MLCK gene and is effective to direct cleavage
specifically of said RNA by RNA interference.
20. A method according to claim 19, wherein the nucleic acid is a
siNA. wherein (a) each strand of said siNA molecule is about 19 to
about 25 nucleotides in length; and (b) one strand of said siNA
molecule comprises a region having a nucleotide sequence having
sufficient complementarity to the MLCK RNA for the siNA molecule to
direct cleavage of the MLCK RNA via RNA interference.
21. A method according to claim 20, wherein the siNA specifically
cleaves MLCK-L RNA and substantially does not cleave MLCK-S
RNA.
22. A method according to claim 21, wherein the siNA is a
siRNA.
23. A method according to claim 22, wherein the siRNA comprises a
region having the sequence of SEQ ID NO:3.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims full right of priority and is a
continuation-in-part application of U.S. Provisional Application
Ser. No. 60/841,000 filed on 30 Aug. 2006 which is herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] In various aspects and embodiments the invention relates
generally to the field of gene expression and its control, to
reagents and methods therefore, and to the treatment of disorders
and diseases thereby. Various embodiments relate to regulation of
MLCK and to interfering nucleic acids therefore. Various
embodiments relate to down-regulation of MLCK-L expression without
concomitant down regulation of MLCK-S, to reagents and methods
therefore, and to the treatment of disorders and diseases
thereby.
BACKGROUND
[0003] Increased lung vascular permeability is a hallmark of acute
respiratory distress syndrome (ARDS). It has been established that
endothelium regulates passage of plasma proteins and other
macromolecules across the vessel wall and performs the vital task
of maintaining the integrity of the alveolar-capillary barrier. See
Mehta and Malik, Physiol. Rev., January 2006; 86(1):279-367.
Endothelial cells regulate the barrier properties of the
microvessel wall by altering their shape, which was earlier
described as "cell rounding" by Majno and Palade (1961). See Majno
et al., J. Biophys. Biochem. Cytol., December, 1961: 11:571-605.
Cell shape change occurs as a result of actinomyosin-based
endothelial cell contraction, requiring myosin light chain (MLC)
phosphorylation. See Mehta and Malik, Physiol. Rev., January 2006;
86(0:279-367. The phosphorylation of MLC is catalyzed by activated
myosin light chain kinase (MLCK) in the presence of ionized
intracellular calcium and calmodulin..sup.3 Several studies using
either constitutively active MLCK or pharmacological inhibitors of
MLCK have shown that MLCK-induced MLC phosphorylation plays an
essential role in regulating endothelial permeability in vitro and
in vivo..sup.3-10 In addition, studies indicate that increased MLCK
activity and MLC phosphorylation in endothelial cell monolayers are
required for transendothelial polymorphonuclear ("PMN") migration
elicited by chemotactic agents..sup.11, 12 Importantly, recent gene
expression profiling studies from several acute lung injury
patients have identified SNPs (single-nucleotide polymorphisms) in
the MLCK gene (MYLK) that may be associated with ARDS. MLCK is thus
indicated to be a potential therapeutic target for treating
ARDS..sup.13, 14
[0004] Interestingly, primary endothelial cells are known to
express the 210 kDa long isoform of MLCK referred to as endothelial
MLCK-L and the well-known 130 kDa smooth muscle MLCK isoform
(MLCK-S, as used herein)..sup.9, 15-17 These isoforms are encoded
from a single gene on chromosome 3. Structurally, MLCK-L contains
all the domains present in the smooth muscle isoform, but in
addition, has a unique 922-amino acid N-terminal domain containing
consensus sites for phosphorylation by multiple protein kinases,
including cAMP-dependent protein kinase A (PKA), PKC, PAK, Src, and
Ca.sup.2+/CaM-dependent protein kinase II..sup.1, 3. The N-terminus
of MLCK-L has been shown to interact with Src..sup.3 Macrophage
inhibitory factor.sup.18 and microtubules.sup.19 also bind with
high affinity to the N-terminus of MLCK-L. Thus, regulation of
MLCK-L by serine and tyrosine kinases, and its complexation with
multiple proteins indicate that many signaling pathways may exert
control on endothelial permeability by converging to MLCK-L.
However, previous studies using approaches which target the kinase
activity of MLCK without isoform specificity have not been clear in
identifying the individual role of MLCK-L in regulating endothelial
barrier function. In accordance with various aspects and
embodiments of the invention herein disclosed an siRNA sequence
that specifically "knocks down" MLCK-L in cultured cells was
developed and used in a recently developed strain of mice lacking
MLCK-L (MLCK210.sup.-/- mice).sup.6 to show the importance of
MLCK210 in the mechanism of increased lung vascular permeability. A
thrombin or a PAR-1 peptide agonist was used to elicit an
endothelial permeability response because PAR-1 receptor agonists
are known to increase endothelial permeability by actinomyosin
induced contraction downstream of G-protein-coupled
proteinase-activated receptor-1 (PAR-1) in endothelial monolayer as
well as in vivo models. The results show that, in accordance with
the invention. MLCK-L is a key effector mediating the PAR-1-induced
increase in lung vascular permeability in part through
phosphorylation of MLC and MLCK is involved in regulation of
calcium entry via a previously undescribed interaction with store
operated channels.
SUMMARY
[0005] In various aspects and embodiments the invention provides
new nucleic acid molecules which down regulate expression and/or
stability of MLCK-L. In various aspects and embodiments the
invention provides compounds and methods which are useful in
molecular investigations of MLCK and, additionally, in the
diagnosis, prevention, and therapy of tissue inflammation and
angiogenesis. In embodiments the compounds are stable nucleic acid
agents which may be used to knockdown or down regulate MLCK-L. In
embodiments the nucleic acid agent is a siRNA. In embodiments, the
nucleic acids are modified to adjust for single-nucleotide
polymorphisms which may be reflected in the targeted DNA or RNA
molecule(s).
[0006] Embodiments of the invention provide a double stranded short
interfering nucleic acid (siNA) molecule that directs cleavage of a
myosin light chain kinase (MLCK) RNA, wherein: (a) each strand of
said siNA molecule is about 19 to about 25 nucleotides in length;
and (b) one strand of said siNA molecule comprises a region having
a nucleotide sequence having sufficient complementarity to the MLCK
RNA for the siNA molecule to direct cleavage of the MLCK RNA via
RNA interference. In embodiments the double-stranded siNA is a
siRNA. In embodiments the siNA cleaves MLCK-L RNA more efficiently
than MLCK-S RNA. In embodiments the siNA cleaves MLCK-L RNA at
least twice as efficiently as it cleaves MLCK-S RNA. In embodiments
the siNA specifically cleaves MLCK-L RNA and substantially does not
cleave MLCK-S RNA. In embodiments, the siRNA comprises a region
having the sequence of SEQ ID NO:3. In embodiments, the siNA is a
siRNA.
[0007] Embodiments of the invention provide a method for modulating
MLCK expression in a cell, comprising introducing into a cell a
double stranded siNA that cleaves MLCK RNA, wherein (a) each strand
of said siNA molecule is about 19 to about 25 nucleotides in
length; and (b). one strand of said siNA molecule comprises a
region having a nucleotide sequence having sufficient
complementarity to the MLCK RNA for the siNA molecule to direct
cleavage of the MLCK RNA via RNA interference. In embodiments the
siNA specifically cleaves MLCK-L RNA and substantially does not
cleave MLCK-S RNA. In embodiments, the siNA comprises a region
having the sequence of SEQ ID NO:3. In embodiments the siNA is a
siRNA.
[0008] Embodiments of the invention provide a method for modulating
tissue inflammation of a patient suffering therefrom, comprising:
administering to a patient suffering from tissue inflammation a
nucleic acid in an amount and by a route effective to modulate said
tissue inflammation, wherein said nucleic acid comprises a region
complementary to an RNA encoded by an MLCK gene and is effective to
direct cleavage specifically of said RNA by RNA interference. In
embodiments the nucleic acid is a double stranded siNA. In
embodiment the nucleic acid is a double stranded siNA that cleaves
MLCK RNA. In embodiment each strand of said siNA molecule is about
19 to about 25 nucleotides in length; and one strand of said siNA
molecule comprises a region having a nucleotide sequence having
sufficient complementarity to the MLCK RNA for the siNA molecule to
direct cleavage of the MLCK RNA via RNA interference. In
embodiments the siNA specifically cleaves MLCK-L RNA and
substantially does not cleave MLCK-S RNA. In embodiments, the siNA
comprises a region having the sequence of SEQ ID NO:3. In
embodiments, the siNA is a siRNA.
[0009] Embodiments of the invention provide a method for treating
asthma in a patient suffering therefrom, comprising: administering
to a patient suffering from asthma a nucleic acid in an amount and
by a route effective to treat asthma, wherein said nucleic acid
comprises a region complementary to an RNA encoded by an MLCK gene
and is effective to direct cleavage specifically of said RNA by RNA
interference. In embodiments the nucleic acid is a siNA. In
embodiments the nucleic acid is a siNA that specifically cleaves
MLCK-L RNA and substantially does not cleave MLCK-S RNA. In
embodiments the nucleic acid comprises a region having the sequence
of SEQ ID NO:3. In embodiments the nucleic acid is a siNA. In
embodiments the nucleic acid is a siRNA.
[0010] In various embodiments the invention provides methods for
modulating expression of genes in a patient comprising:
administering to a patient a nucleic acid complementary to a target
3'UTR mRNA encoded by a gene expressed in the endothelium in an
amount and by a route effective for modulating the activity of said
mRNA in said patient. In embodiments in this regard the nucleic
acid is a siNA. In embodiments the nucleic acid specifically
cleaves MLCK RNAs. In embodiments the nucleic acid specifically
cleaves MLCK-L RNA and substantially does not cleave MLCK-S RNA. In
embodiments the nucleic acid is a siNA. In embodiments the nucleic
acid is a siRNA. In embodiments the nucleic acid comprises a region
complementary to a region of an MLCK RNA. In embodiments the region
has greater complementary to MLCK-L than to MLCK-S RNA. In
embodiments the region is complementary to all or a part of the
sequence of nucleotides 1428 to 1634 of the sequence of human
MLCK-L RNA, as set out in SEQ ID NO. 4. In embodiments the nucleic
acid directs cleavage by RNA interference of MLCK specifically in
non-smooth muscle cells but substantially not in smooth muscle
cells. In embodiments the nucleic acid comprises a region having
the sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
[0011] In embodiments the nucleic acid is effective to treat
Sepsis, Acute Respiratory Distress Syndrome, Trauma, Inflammation
and/or Asthma.
[0012] RNA interference refers to the process of sequence-specific
post-transcriptional gene silencing in animals mediated by short
interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33;
Fire et al., 1998, Nature, 391, 806; Hamilton et al., 1999,
Science, 286, 950-951; Lin et al., 1999, Nature, 402, 128-129;
Sharp, 1999, Genes & Dev., 13:139-141; and Strauss, 1999,
Science, 286, 886). The corresponding process in plants (Heifetz et
al., International PCT Publication No. WO 99/61631) is commonly
referred to as post-transcriptional gene silencing or RNA silencing
and is also referred to as quelling in fungi. The process of
post-transcriptional gene silencing is thought to be an
evolutionarily-conserved cellular defense mechanism used to prevent
the expression of foreign genes and is commonly shared by diverse
flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such
protection from foreign gene expression may have evolved in
response to the production of double-stranded RNAs (dsRNAs) derived
from viral infection or from the random integration of transposon
elements into a host genome via a cellular response that
specifically destroys homologous single-stranded RNA or viral
genomic RNA. The presence of dsRNA in cells triggers the RNAi
response through a mechanism that has yet to be fully
characterized. This mechanism appears to be different from other
known mechanisms involving double stranded RNA-specific
ribonucleases, such as the interferon response that results from
dsRNA-mediated activation of protein kinase PKR and
2',5'-oligoadenylate synthetase resulting in non-specific cleavage
of mRNA by ribonuclease L (see for example U.S. Pat. Nos.
6,107,094; 5,898,031; Clemens et al., 1997, J. Interferon &
Cytokine Res., 17, 503-524; Adah et al., 2001, Curr. Med. Chem., 8,
1189).
[0013] In particular, the instant invention features small nucleic
acid molecules, such as short interfering nucleic acid (siNA),
short interfering RNA (siRNA), double-stranded RNA (dsRNA),
micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and
methods used to modulate the expression of MLCK genes.
[0014] A siNA of the invention can be unmodified or
chemically-modified. A siNA of the instant invention can be
chemically synthesized, expressed from a vector or enzymatically
synthesized. The instant invention also features various
chemically-modified synthetic short interfering nucleic acid (siNA)
molecules capable of modulating MLCK gene expression or activity in
cells by RNA interference (RNAi). The use of chemically-modified
siNA improves various properties of native siNA molecules through,
for example, increased resistance to nuclease degradation in vivo
and/or through improved cellular uptake. Furthermore, siNA having
multiple chemical modifications may retain its RNAi activity. The
siNA molecules of the instant invention provide useful reagents and
methods for a variety of therapeutic, diagnostic, target
validation, genomic discovery, genetic engineering, and
pharmacogenomic applications.
FIGURES
[0015] FIG. 1. Knockdown of endogenous expression of MLCK-L with
siRNA
[0016] (A) After 48 hr, cells transfected with MLCK-L siRNA
sequences (1-3) or control cells were lysed and Western blotted
using MLCK antibody to detect protein expression. (B) Plot shows
mean.+-.SE of percent reduction in MLCK-L expression after
knockdown normalized against MLCK-L expression in control cells. *
indicates decrease in MLCK-L expression in seq3 siRNA transfected
cells (p<0.05).
TABLE-US-00001 Sequence 1 corresponds to: NNGGACUGCGCUGUUAUUGAG.
(SEQ ID NO: 1) Sequence 2 corresponds to: NNGUGGAAAGGCUUGCCGUGA.
(SEQ ID NO:2) Sequence 3 corresponds to: NNUGGGCAGCCCAUCCAGUAC.
(SEQ ID NO:3)
[0017] FIG. 2. Effect of MLCK-L knockdown on MLC phosphorylation in
response to thrombin
[0018] (A) Western blot showing MLC phosphorylation in response to
thrombin in cells transfected with control (cont si) or MLCK-L
(Si-MLCK-L) siRNA. Cells were stimulated with thrombin 48 hr post
transfection and lysed. Lysates were Western blotted with
phospho-MLC (top panel) or pan MLC Abs (bottom panel) to determine
MLC phosphorylation. (B) Scatter plot showing mean.+-.SEM of MLC
phosphorylation normalized to total MLC in control or MLCK-L siRNA
transfected monolayers (n=3).
[0019] FIG. 3. Effect of MLCK-L knockdown on thrombin-induced RhoA
signaling
[0020] (A) RhoA activity after 2 min thrombin stimulation of
control or MLCK-L siRNA transfected HPAE cells. RhoA activation is
measured by the increased amount of GTP-bound RhoA (top) compared
to total amount of RhoA in whole cell lysates (bottom). (B) MYPT
phosphorylation induced by thrombin in control (cont Si) or MLCK-L
(Si MLCK) siRNA transfected HPAE cells. Bottom panel shows equal
protein loading as analyzed by Western blotting with actin Ab. (C),
scatter plot showing mean.+-.SEM of MYPT phosphorylation normalized
to actin in control or MLCK-L siRNA transfected monolayers
(n=3).
[0021] FIG. 4. Effect of MLCK-L knockdown on store operated calcium
entry
[0022] Ratiometric measurements of [Ca.sup.2+].sub.i during
extracellular Ca.sup.2+ depletion-repletion conditions after
depletion of stores with thrombin (A-B) or thapsigargin (C-D) in
cells transfected with control (cont Si; solid line) or MLCK-L
siRNA (Si MLCK; broken line). Measurements were made 48 hr post
transfection after loading HPAE cell monolayer with Fura 2-AM for
15 min. Each representative tracing is the average response of
30-40 cells, and experiments were repeated three times. B and D,
plot shows mean.+-.SEM of [Ca.sup.2+].sub.i following store
depletion or Ca.sup.2+ entry in two groups (n=5). * indicates
reduced Ca.sup.2+ entry in MLCK-L knockdown cells compared to cells
transfected with control siRNA transfected cells (P<0.05).
[0023] FIG. 5. MLCK-L knockdown effect on throbin stimulated
aderens junction disassembly
[0024] Cells transfected with control (Cont Si) or MLCK-L (Si
MLCK-L) siRNA were stimulated with thrombin 48 hr post transfection
and fixed followed by staining with VE-cadherin and Alexa-labeled
secondary Ab to determine adherens junction dis-assembly by
confocal imaging.
[0025] FIG. 6. MLCK-L knockdown effect on thrombin stimulated
barrier dysfunction
[0026] Transendothelial electrical resistance (TER) measurements in
HPAE cells transfected with control (cont Si) or MLCK-L (Si MLCK-L)
siRNA. Data represent mean.+-.SE from multiple experiments
(n=5).
[0027] FIG. 7. Effects of PAR-1 agonist peptide on pulmonary
microvessel liquid permeability (K.sub.fc) in lungs isolated from
Wt or MLCK-L knockout mice
[0028] (A) Immunoblot of lung homogenate with MLCK antibody shows
absence of MLCK-L 210 in MLCK.sup.-/- mice lungs but expression of
130 KD smooth muscle MLCK isoform is not affected. (B) After
attaining isogravimetric conditions in the lungs, venous pressure
was raised to 10 cm H.sub.2O to determine changes in K.sub.f,c.
Results are mean.+-.SE of 4 experiments. C, PAR-1 peptide induced
vasoconstrictor response in lungs isolated from Wt or MLCK.sup.-/-
mice.
[0029] FIG. 8. Western blot showing unphosphorylated (UP) and
phosphorylated foul) (P) of mouse lung myosin light chain (MLC)
[0030] WT (wild type) or MLCK-L.sup.-/- lung preparations were
preperfused for 7 min with PAR-1 peptide and MLC phosphorylation
was determined by Western blotting with anti-MLCK antibody
following urea gel electrophoresis.
[0031] FIG. 9. Inhibition of eye inflammation in mouse model
lacking the MLCK gene
[0032] Numbers of inflammatory cells in subconjunctival areas in
paraffin eye sections were counted. The value was normalized to the
number of inflammatory cells per unit area (2500 .mu.m.sup.2)
underneath the conjunctival epithelium. For each treatment at least
4 mice were used.
[0033] FIG. 10. MLCK-L siRNA prevents LPS-induced lung injury
[0034] After 48 hours mice (C56Bl/J) injected with scrambled (Sc)
or MLCK-L siRNA were challenged with PBS or 1 mg/ml LPS through
nebulizer for 45 min. Lungs were harvested after 4 hr of LPS
challenge. Inset: Immunoblot of mouse lung injected with MLCK-L
siRNA shows decrease in MLCK-L protein expression (210 kD). Note:
MLCK-L siRNA had minimal effect on short (130 kD) smooth muscle
MLCK isoform. (A) lung wet- dry-weight ratio in mice receiving Sc
or MLCK-SiRNA. (B) albumin accumulation in mouse lungs receiving Sc
or MLCK-SiRNA. Evans-blue tagged albumin was injected into
vasculature of 30 min before terminating the experiment and
accumulation of Evans-blue was determined. (C-D) Inflammatory cells
accumulation in brochoalveolar lavage of mice receiving Sc or
MLCK-SiRNA. Neutph, neutrophils; Bas, basophils; Eos, eosinophils;
lymph, lymphocytes; macroph, macrophages.
DESCRIPTION
[0035] The present invention relates to compounds and methods which
are useful in molecular investigations of MLCK, and, in the
diagnosis, prevention, and therapy of tissue inflammation and
angiogenesis. These compounds are stable nucleic acid agents which
may be used to knockdown or down regulate MLCK-L. An example of one
such nucleic acid agent is a siRNA as herein described.
Glossary
[0036] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices, and materials are as
described.
[0037] As used herein, particularly in the claims, the words "a"
and "an" mean the same as the phrases "one and more than one" and
"at least one" and explicitly are not limited to the singular, or
to just one. For example, "a host cell" refers to at least one host
cell and includes more than one host cell.
[0038] As used herein, particularly in the claims, the word "the"
includes the plural as well as the singular and refers to "one or
more than one" and "at least one." For example, "the siRNA" refers
to at least one siRNA, and includes one and more than one
siRNA.
[0039] The word "specifically" as used herein in, particularly as
to the phrase "specifically cleaves MLCK-L RNA," means that at
least 50% of all the RNAs that are cleaved are MLCK-L RNAs.
[0040] The term "substantially" as used herein, in particular in
the phrase "substantially does not cleave" means cleavage does not
occur or is inefficient. For instance, in a method of treatment the
phase means that cleavage, to the extent it occurs at all, does not
engender deleterious and/or adverse effects on a patient.
[0041] The term "more efficiently" as used herein means greater
than.
[0042] The term "siRNA" refers to a small interfering RNA(s), which
also has been referred to in the art as short interfering RNA and
silencing RNA, among others. siRNAs generally are described as
relatively short, often 20-25 nucleotide-long, double-stranded RNA
molecules that are involved in RNA interference (RNAi) pathway(s).
Generally, siRNAs are, in part, complementary to specific mRNAs and
mediate their down regulation (hence, "interfering"). siRNAs thus
can he used for down regulating the expression of specific genes
and gene function in cells and organisms. siRNAs also play a role
in related pathways. The general structure of most naturally
occurring siRNAs is well established. Generally, siRNAs are short
double-stranded RNAs, usually 21 nucleotides long, with two
nucleotides single stranded "overhangs" on the 3 of each strand.
Each strand has a 5' phosphate group and a 3' hydroxyl (--OH)
group. In vivo, the structure results from processing by the enzyme
"dicer," which enzymatically converts relatively long dsRNAs and
relatively small hairpin RNAs into siRNAs.
[0043] The term siNA refers to a nucleic acid that acts like a
siRNA, as described herein, but may be other than an RNA, such as a
DNA, a hybrid RNA:DNA or the like. siNAs function like siRNAs to
down regulate expression of gene products. See siRNA.
[0044] The term "RNA interference" which also has been called "RNA
mediated interference" refers to the cellular processes by which
RNA (such as siRNAs) down regulate expression of genes; i.e., down
regulate or extinguish the expression of gene functions, such as
the synthesis of a protein encoded by a gene. Typically,
double-stranded ribonucleic acid inhibits the expression of genes
with complementary nucleotide sequences. RNA interference pathways
are conserved in most eukaryotic organisms. It is initiated by the
enzyme dicer, which cleaves RNA, particularly double-stranded RNA,
into short double-stranded fragments 20-25 base pairs long. One
strand of the double-stranded RNA (called the "guide strand") is
part of a complex of proteins called the RNA-induced silencing
complex (RISC). The thus incorporated guide strand serves as a
recognition sequence for binding of the RISC to nucleic acids with
complementary sequences. Binding by RISC to complementary nucleic
acids results in their being "silenced." The best studied silencing
is the binding of RISCs to RNAs resulting in post-transcriptional
gene silencing. Regardless of mechanism, interfering nucleic acids
and RNA interference result in down regulation of the target gene
or genes that are complementary (in pertinent part) to the guide
strand.
[0045] A polynucleotide can be delivered to a cell to express an
exogenous nucleotide sequence, to inhibit, eliminate, augment, or
alter expression of an endogenous nucleotide sequence, or to affect
a specific physiological characteristic not naturally associated
with the cell. The polynucleotide can be a sequence whose presence
or expression in a cell alters the expression or function of
cellular genes or RNA. A delivered polynucleotide can stay within
the cytoplasm or nucleus apart from the endogenous genetic
material. Alternatively, DNA can recombine with (become apart of)
the endogenous genetic material. Recombination can cause DNA to be
inserted into chromosomal DNA by either homologous or
non-homologous recombination.
[0046] A polynucleotide-based gene expression inhibitor comprises
any polynucleotide containing a sequence whose presence or
expression in a cell causes the degradation of or inhibits the
function, transcription, or translation of a gene in a
sequence-specific manner. Polynucleotide-based expression
inhibitors may be selected from the group comprising: siRNA,
microRNA, interfering RNA or RNAi, dsRNA, ribozymes, antisense
polynucleotides, and DNA expression cassettes encoding siRNA,
microRNA, dsRNA, ribozymes or antisense nucleic acids. SiRNA
comprises a double stranded structure typically containing 15 to 50
base pairs and preferably 19 to 25 base pairs and having a
nucleotide sequence identical or nearly identical to an expressed
target gene or RNA within the cell. An siRNA may be composed of two
annealed polynucleotides or a single polynucleotide that forms a
hairpin structure. MicroRNAs (miRNAs) are small noncoding
polynucleotides, about 22 nucleotides long, that direct destruction
or translational repression of their mRNA targets. Antisense
polynucleotides comprise a sequence that is complimentary to a gene
or mRNA. Antisense polynucleotides include, but are not limited to:
morpholinos, 2'-O-methyl polynucleotides, DNA, RNA and the like.
The polynucleotide-based expression inhibitor may be polymerized in
vitro, recombinant, contain chimeric sequences, or derivatives of
these groups. The polynucleotide-based expression inhibitor may
contain ribonucleotides, deoxyribonucleotides, synthetic
nucleotides, or any suitable combination such that the target RNA
and/or gene is inhibited.
[0047] Polynucleotides may contain an expression cassette coded to
express a whole or partial protein, or RNA. An expression cassette
refers to a natural or recombinantly produced polynucleotide that
is capable of expressing a sequence. The cassette contains the
coding region of the gene of interest along with any other
sequences that affect expression of the sequence of interest. An
expression cassette typically includes a promoter (allowing
transcription initiation), and a transcribed sequence. Optionally,
the expression cassette may include, but is not limited to,
transcriptional enhancers, non-coding sequences, splicing signals,
transcription termination signals, and polyadenylation signals. An
RNA expression cassette typically includes a translation initiation
codon (allowing translation initiation), and a sequence encoding
one or more proteins. Optionally, the expression cassette may
include, but is not limited to, translation termination signals, a
polyadenosine sequence, internal ribosome entry sites (IRES), and
non-coding sequences. The polynucleotide may contain sequences that
do not serve a specific function in the target cell but are used in
the generation of the polynucleotide. Such sequences include, but
are not limited to, sequences required for replication or selection
of the polynucleotide in a host organism.
[0048] A polynucleotide can be delivered to a cell to study gene
function. Delivery of a polynucleotide to a cell can also have
potential clinical applications. Clinical applications include
treatment of muscle disorders or injury, circulatory disorders,
endocrine disorders, immune modulation and vaccination, and
metabolic disorders (Baumgartner et al. 1998, Blau et al. 1995,
Svensson et al. 1996, Baumgartner et al. 1998, Vale et al. 2001,
Simovic et al. 2001).
[0049] A transfection agent, or transfection reagent, or delivery
vehicle is a compound or compounds that bind(s) to or complex(es)
with oligonucleotides and polynucleotides, and enhances their entry
into cells. Examples of transfection reagents include, but are not
limited to, cationic liposomes and lipids, polyamines, calcium
phosphate precipitates, polycations, histone proteins,
polyethylenimine, polylysine, and polyampholyte complexes. For
delivery in vivo, complexes made with sub-neutralizing amounts of
cationic transfection agent may be preferred. Non-viral vectors
include protein and polymer complexes (polyplexes), lipids and
liposomes (lipoplexes), combinations of polymers and lipids
(lipopolyplexes), and multilayered and recharged particles.
Transfection agents may also condense nucleic acids. Transfection
agents may also be used to associate functional groups with a
polynucleotide. Functional groups include cell targeting moieties,
cell receptor ligands, nuclear localization signals, compounds that
enhance release of contents from endosomes or other intracellular
vesicles (such as membrane active compounds), and other compounds
that alter the behavior or interactions of the compound or complex
to which they are attached (interaction modifiers).
[0050] The term "naked nucleic acids" indicates that the nucleic
acids are not associated with a transfection reagent or other
delivery vehicle that is required for the nucleic acid to be
delivered to a target cell.
[0051] "Inhibit" or "down-regulate" means that the expression of
the gene, or level of RNAs or equivalent RNAs encoding one or more
proteins or isoforms, or activity of one or more proteins, such as
the MLCK-L or MLCK-S forms, is reduced below that observed in the
absence of the nucleic acid molecules of the invention. In one
embodiment, inhibition or down-regulation with the presently
described nucleic acid molecules preferably is below that level
observed in the presence of an enzymatically inactive or attenuated
molecule that is able to bind to the same site on the target RNA,
but is unable to cleave that RNA. In another embodiment, inhibition
or down-regulation with antisense oligonucleotides is preferably
below that level observed in the presence of, for example, an
oligonucleotide with scrambled sequence or with mismatches. In
another embodiment, inhibition or down-regulation of MLCK with the
nucleic acid molecule of the instant invention is greater in the
presence of the nucleic acid molecule than in its absence.
[0052] By "up-regulate" is meant that the expression of the gene,
or level of RNAs or equivalent RNAs encoding one or more proteins
or isoforms, or activity of one or more proteins, such as MLCK-L or
MLCK-S, is greater than that observed in the absence of the nucleic
acid molecules of the invention. For example, the expression of a
gene, such as the MLCK gene, can be increased in order to treat,
prevent, ameliorate, or modulate a pathological condition caused or
exacerbated by an absence or low level of gene expression.
[0053] By "modulate" is meant that the expression of the gene, or
level of RNAs or equivalent RNAs encoding one or more protein
subunits, or activity of one or more proteins or protein isoforms
is up-regulated or down-regulated, such that the expression, level,
or activity is greater than or less than that observed in the
absence of the nucleic acid molecules of the invention.
[0054] By "gene" it is meant a nucleic acid that encodes an RNA,
for example, nucleic acid sequences including but not limited to
structural genes encoding a polypeptide.
[0055] "Complementarity" refers to the ability of a nucleic acid to
form hydrogen bond(s) with another RNA sequence by either
traditional Watson-Crick or other non-traditional types. In
reference to the nucleic molecules of the present invention, the
binding free energy for a nucleic acid molecule with its target or
complementary sequence is sufficient to allow the relevant function
of the nucleic acid to proceed, e.g., enzymatic nucleic acid
cleavage, antisense or triple helix inhibition. Determination of
binding free energies for nucleic acid molecules is well known in
the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII
pp. 123 (33; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373
9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783 3785). A
percent complementarity indicates the percentage of contiguous
residues in a nucleic acid molecule which can form hydrogen bonds
(e.g., Watson-Crick base pairing) with a second nucleic acid
sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%,
80%, 90%, and 100% complementary). "Perfectly complementary" means
that all the contiguous residues of a nucleic acid sequence will
hydrogen bond with the same number of contiguous residues in a
second nucleic acid sequence.
[0056] By "RNA" is meant a molecule comprising at least one
ribonucleotide residue. By "ribonucleotide" or "2'-OH" is meant a
nucleotide with a hydroxyl group at the 2' position of a
JD-ribo-furanose moiety.
[0057] Nucleic Acid Modification.
[0058] Chemically synthesizing nucleic acid molecules with
modifications (base, sugar and/or phosphate) that prevent their
degradation by serum ribonucleases can increase their potency (see
e.g., Eckstein et a)., International Publication No. WO 92/07065;
Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991, Science
253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17,
334; Usman et al., International Publication No. WO 93/15187; and
Rossi et al., International Publication No. WO 91/03162; Sproat,
U.S. Pat. No. 5,334,711; and Burgin et al., supra; all of these
describe various chemical modifications that can be made to the
base, phosphate and/or sugar moieties of the nucleic acid molecules
herein). Modifications which enhance their efficacy in cells, and
removal of bases from nucleic acid molecules to shorten
oligonucleotide synthesis times and reduce chemical requirements
are desired. (All these publications are hereby incorporated by
reference herein).
[0059] There are several examples in the art describing sugar, base
and phosphate modifications that can be introduced into nucleic
acid molecules with significant enhancement in their nuclease
stability and efficacy. For example, oligonucleotides are modified
to enhance stability and/or enhance biological activity by
modification with nuclease resistant groups, for example, 2'-amino,
2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'-H, nucleotide base
modifications (for a review see Usman and Cedergren, 1992, TIBS.
17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163;
Burgin et al., 1996, Biochemistry, 35, 14090). Sugar modification
of nucleic acid molecules have been extensively described in the
art (see Eckstein et al., International Publication PCT No. WO
92/07065; Perrault et al. Nature, 1990, 344, 565 568; Pieken et al.
Science, 1991, 253, 314 317; Usman and Cedergren, Trends in
Biochem. Sci., 1992, 17, 334 339; Usman et al. International
Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711
and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman
et al., International PCT publication No. WO 97/26270; Beigelman et
al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No.
5,627,053; Woolf et al., International PCT Publication No. WO
98/13526; Thompson et al., U.S. Ser. No. 60/082,404 which was filed
on Apr. 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39,
1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic acid Sciences),
48, 39 55; Velma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99
134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999 2010;
all of the references are hereby incorporated in their totality by
reference herein). Such publications describe general methods and
strategies to determine the location of incorporation of sugar,
base and/or phosphate modifications and the like into ribozymes
without inhibiting catalysis, and are incorporated by reference
herein. In view of such teachings, similar modifications can be
used as described herein to modify the nucleic acid molecules of
the instant invention.
[0060] While chemical modification of oligonucleotide
internucleotide linkages with phosphorothioate, phosphorothioate,
and/or 5'-methylphosphonate linkages improves stability, too many
of these modifications can cause some toxicity. Therefore when
designing nucleic acid molecules the amount of these
internucleotide linkages should be minimized. The reduction in the
concentration of these linkages should lower toxicity resulting in
increased efficacy and higher specificity of these molecules.
[0061] Nucleic acid molecules having chemical modifications that
maintain or enhance activity are provided. Such nucleic acid is
also generally more resistant to nucleases than unmodified nucleic
acid. Thus, in a cell and/or in vivo the activity can not be
significantly lowered. Therapeutic nucleic acid molecules delivered
exogenously are optimally stable within cells until translation of
the target RNA has been inhibited long enough to reduce the levels
of the undesirable protein. This period of time varies between
hours to days depending upon the disease state. Nucleic acid
molecules are preferably resistant to nucleases in order to
function as effective intracellular therapeutic agents.
Improvements in the chemical synthesis of RNA and DNA (Wincott et
al., 1995 Nucleic Acids Res. 23, 2677; Caruthers et al., 1992,
Methods in Enzymology 211,3 19 (incorporated by reference herein)
have expanded the ability to modify nucleic acid molecules by
introducing nucleotide modifications to enhance their nuclease
stability as described above.
[0062] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that down-regulates
expression of a MLCK gene, wherein the siNA molecule comprises
about 19 to about 21 base pairs, and wherein each strand of the
siNA molecule comprises one or more chemical modifications. In
another embodiment, one of the strands of the double-stranded siNA
molecule comprises a nucleotide sequence that is complementary to a
nucleotide sequence of a MLCK gene or a portion thereof, and the
second strand of the double-stranded siNA molecule comprises a
nucleotide sequence substantially similar to the nucleotide
sequence or a portion thereof of the targeted gene. In another
embodiment, one of the strands of the double-stranded siNA molecule
comprises a nucleotide sequence that is complementary to a
nucleotide sequence of a MLCK gene or a portion thereof, and the
second strand of the double-stranded siNA molecule comprises a
nucleotide sequence substantially similar to the nucleotide
sequence or a portion thereof of the MLCK gene. In another
embodiment, each strand of the siNA molecule comprises about 19 to
about 23 nucleotides, and each strand comprises at least about 19
nucleotides that are complementary to the nucleotides of the other
strand. The MLCK gene may comprise, for example, the sequence
referred to in Table I, or a sub-sequence thereof.
[0063] In embodiment of the invention the siNA comprises a sequence
that has greater complementarity to MLCK-L than MLCK-S and directs
down regulation of MLCK-L more efficiently than it directs down
regulation of MLCK-S. In embodiments in this regard the siNA
contains a region that is at least 90% identical in sequence to a
region of MLCK-L inRNA but is less than 50% identical to any region
in MLCK-S inRNA. In embodiments the siRNA comprises a region having
a sequence comprised within the MLCK-L sequence from nucleotides
1428 to 1638 (inclusive) in SEQ/ID NO 24 (or its complement). In
embodiments said sequence in MLCK-L is not found in MLCK-S.
TABLE-US-00002 TABLE I (SEQ ID NO: 4) Homo sapiens myosin light
chain 1 ccatggggga tgtgaagctg gttgcctcgt cacacatttc caaaacctcc
ctcagtgtgg polypeptide kinase isoform 1 61 atccctcaag agttgactcc
atgcccctga cagaggcccc tgctttcatt ttgccccctc (MLCK) 121 ggaacctctg
catcaaagaa ggagccaccg ccaagttcga agggcgggtc cggggttacc Accession:
AY424270 181 cagagcccca ggtgacatgg cacagaaacg ggcaacccat caccagcggg
ggccgcttcc 5776 bp mRNA linear Complete 241 tgctggattg cggcatccgg
gggactttca gccttgtgat tcatgctgtc catgaggagg CDS; alternatively
spliced 301 acaggggaaa gtatacctgt gaagccacca atggcagtgg tgctcgccag
gtgacagtgg 361 agttgacagt agaaggaagt tttgcgaagc agcttggtca
gcctgttgtt tccaaaacct 421 taggggatag attttcagct tcagcagtgg
agacccgtcc tagcatctgg ggggagtgcc 481 caccaaagtt tgctaccaag
ctgggccgag ttgtggtcaa agaaggacag atgggacgat 541 tctcctgcaa
gatcactggc cggccccaac cgcaggtcac ctggctcaag ggaaatgttc 601
cactgcagcc gagtgcccgt gtgtctgtgt ctgagaagaa cggcatgcag gttctggaaa
661 tccatggagt caaccaagat gacgtgggag tgtacacgtg cctggtggtg
aacgggtcgg 721 ggaaggcctc gatgtcagct gaactttcca tccaaggttt
ggacagtgcc aataggtcat 781 ttgtgagaga aacaaaagcc accaattcag
atgtcaggaa agaggtgacc aatgtaatct 841 caaaggagtc gaagctggac
agtctggagg ctgcagccaa aagcaagaac tgctccagcc 901 cccagagagg
tggctcccca ccctgggctg caaacagcca gcctcagccc ccaagggagt 961
ccaagctgga gtcatgcaag gactcgccca gaacggcccc gcagaccccg gtccttcaga
1021 agacttccag ctccatcacc ctgcaggccg caagagttca gccggaacca
agagcaccag 1081 gcctgggggt cctatcacct tctggagaag agaggaagag
gccagctcct ccccgtccag 1141 ccaccttccc caccaggcag cctggcctgg
ggagccaaga tgttgtgagc aaggctgcta 1201 acaggagaat ccccatggag
ggccagaggg attcagcatt ccccaaattt gagagcaagc 1261 cccaaagcca
ggaggtcaag gaaaatcaaa ctgtcaagtt cagatgtgaa gtttccggga 1321
ttccaaagcc tgaagtggcc tggttcctgg aaggcacccc cgtgaggaga caggaaggca
1381 gcattgaggt ttatgaaaat gctggctccc attacctctg cctgctgaaa
gcccggacca 1441 gggacagtgg gacatacagc tgcactgctt ccaacgccca
aggccaggtg tcctgtagct 1501 ggaccctcca agtggaaagg cttgccgtga
tggaggtggc cccctccttc tccagtgtcc 1561 tgaaggactg cgctgttatt
gagggccagg attttgtgct gcagtgctcc gtacggggga 1621 ccccagtgcc
ccggatcact tggctgctga atgggcagcc catccagtac gctcgctcca 1681
cctgcgaggc cggcgtggct gagctccaca tccaggatgc cctgccggag gaccatggca
1741 cctacacctg cctagctgag aatgccttgg ggcaggtgtc ctgcagcgcc
tgggtcaccg 1801 tccatgaaaa gaagagtagc aggaagagtg agtaccttct
gcctgtggct cccagcaagc 1861 ccactgcacc catcttcctg cagggcctct
ctgatctcaa agtcatggat ggaagccagg 1921 tcactatgac tgtccaagtg
tcagggaatc caccccctga agtcatctgg ctgcacaatg 1981 ggaatgagat
ccaagagtca gaggacttcc actttgaaca gagaggaact cagcacagcc 2041
tttgtatcca ggaagtgttc ccggaggaca cgggcacgta cacctgcgag gcctggaaca
2101 gcgctggaga ggtccgcacc caggccgtgc tcacggtaca agagcctcac
gatggcaccc 2161 agccctggtt catcagtaag cctcgctcag tgacagcctc
cctgggccag agtgtcctca 2221 tctcctgcgc catagctggt gacccctttc
ctaccgtgca ctggctcaga gatggcaaag 2281 ccctctgcaa agacactggc
cacttcgagg tgcttcagaa tgaggacgtg ttcaccctgg 2341 ttctaaagaa
ggtgcagccc tggcatgccg gccagtatga gatcctgctc aagaaccggg 2401
ttggcgaatg cagttgccag gtgtcactga tgctacagaa cagctctgcc agagcccttc
2461 cacgggggag ggagcctgcc agctgcgagg acctctgtgg tggaggagtt
ggtgctgatg 2521 gtggtggtag tgaccgctat gggtccctga ggcctggctg
gccagcaaga gggcagggtt 2581 ggctagagga ggaagacggc gaggacgtgc
gaggggtgct gaagaggcgc gtggagacga 2641 ggcagcacac tgaggaggcg
atccgccagc aggaggtgga gcagctggac ttccgagacc 2701 tcctggggaa
gaaggtgagt acaaagaccc tatcggaaga cgacctgaag gagatcccag 2761
ccgagcagat ggatttccgt gccaacctgc aacggcaagt gaagccaaag actgtgtctg
2821 aggaagagag gaaggtgcac agcccccagc aggtcgattt tcgctctgtc
ctggccaaga 2881 aggggacttc caagaccccc gtgcctgaga aggtgccacc
gccaaaacct gccaccccgg 2941 attttcgctc agtgctgggt ggcaagaaga
aattaccagc agagaatggc agcagcagtg 3001 ccgagaccct gaatgccaag
gcagtggaga gttccaagcc cctgagcaat gcacagcctt 3061 cagggccctt
gaaacccgtg ggcaacgcca agcctgctga gaccctgaag ccaatgggca 3121
acgccaagcc tgccgagacc ctgaagccca tgggcaatgc caagcctgat gagaacctga
3181 aatccgctag caaagaagaa ctcaagaaag acgttaagaa tgatgtgaac
tgcaagagag 3241 gccatgcagg gaccacagat aatgaaaaga gatcagagag
ccaggggaca gccccagcct 3301 tcaagcagaa gctgcaagat gttcatgtgg
cagagggcaa gaagctgctg ctccagtgcc 3361 aggtgtcttc tgacccccca
gccaccatca tctggacgct gaacggaaag accctcaaga 3421 ccaccaagtt
catcatcctc tcccaggaag gctcactctg ctccgtctcc atcgagaagg 3481
cactgcctga ggacagaggc ttatacaagt gtgtagccaa gaatgacgct ggccaggcgg
3541 agtgctcctg ccaagtcact gtggatgatg ctccagccag tgagaacacc
aaggccccag 3601 agatgaaatc ccggaggccc aagagctctc ttcctcccgt
gctaggaact gagagtgatg 3661 cgactgtgaa aaagaaacct gcccccaaga
cacctccgaa ggcagcaatg ccccctcaga 3721 tcatccagtt ccctgaggac
cagaaggtac gcgcaggaga gtcagtggag ctgtttggca 3781 aagtgacagg
cactcagccc atcacctgta cctggatgaa gttccgaaag cagatccagg 3841
aaagcgagca catgaaggtg gagaacagcg agaatggcag caagctcacc atcctggccg
3901 cgcgccagga gcactgcggc tgctacacac tgctggtgga gaacaagctg
ggcagcaggc 3961 aggcccaggt caacctcact gtcgtggata agccagaccc
cccagctggc acaccttgtg 4021 cctctgacat tcggagctcc tcactgaccc
tgtcctggta tggctcctca tatgatgggg 4081 gcagtgctgt acagtcctac
agcatcgaga tctgggactc agccaacaag acgtggaagg 4141 aactagccac
atgccgcagc acctctttca acgtccagga cctgctgcct gaccacgaat 4201
ataagttccg tgtacgtgca atcaacgtgt atggaaccag tgagccaagc caggagtctg
4261 aactcacaac ggtaggagag aaacctgaag agccgaagga tgaagtggag
gtgtcagatg 4321 atgatgagaa ggagcccgag gttgattacc ggacagtgac
aatcaatact gaacaaaaag 4381 tatctgactt ctacgacatt gaggagagat
taggatctgg gaaatttgga caggtctttc 4441 gacttgtaga aaagaaaact
cgaaaagtct gggcagggaa gttcttcaag gcatattcag 4501 caaaagagaa
agagaatatc cggcaggaga ttagcatcat gaactgcctc caccccccta 4561
agctggtcca gtgtgtggat gcctttgaag aaaaggccaa catcgtcatg gtcctggaga
4621 tcgtgtcagg aggggagctg tttgagcgca tcattgacga ggactttgag
ctgacggagc 4681 gtgagtgcat caagtacatg cggcagatct cggagggagt
ggagtacatc cacaagcagg
[0064] Use of the nucleic acid-based molecules of the invention can
lead to better treatment of the disease progression by affording
the possibility of combination therapies (e.g., multiple antisense
or enzymatic nucleic acid molecules targeted to different genes,
nucleic acid molecules coupled with known small molecule
inhibitors, or intermittent treatment with combinations of
molecules (including different motifs) and/or other chemical or
biological molecules). The treatment of subjects with nucleic acid
molecules can also include combinations of different types of
nucleic acid molecules.
[0065] Therapeutic nucleic acid molecules (e.g., enzymatic nucleic
acid molecules and antisense nucleic acid molecules) delivered
exogenously are optimally stable within cells until translation of
the target RNA has been inhibited long enough to reduce the levels
of the undesirable protein. This period of time varies between
hours to days depending upon the disease state. These nucleic acid
molecules should be resistant to nucleases in order to function as
effective intracellular therapeutic agents. Improvements in the
chemical synthesis of nucleic acid molecules described in the
instant invention and in the art have expanded the ability to
modify nucleic acid molecules by introducing nucleotide
modifications to enhance their nuclease stability as described
above.
[0066] In one embodiment, nucleic acid catalysts having chemical
modifications that maintain or enhance enzymatic activity are
provided. Such nucleic acids are also generally more resistant to
nucleases than unmodified nucleic acid. Thus, in a cell and/or in
vivo the activity of the nucleic acid cannot be significantly
lowered. As exemplified herein such enzymatic nucleic acids are
useful in a cell and/or in vivo even if activity over all is
reduced about 10 fold (Burgin et al., 1996, Biochemistry, 35,
14090). Such enzymatic nucleic acids herein are said to "maintain"
the enzymatic activity of an all RNA ribozyme or all DNA
DNAzyme.
[0067] In another aspect of the invention, vectors, preferably
expression vectors, contain nucleic acids encoding one or more
siNAs, for example, miRNAs. As used herein, the term "vector"
refers to a nucleic acid molecule capable of transporting another
nucleic acid to which it has been linked. One type of a vector is a
"plasmid," which refers to a circular double stranded DNA loop into
which additional DNA segments can be ligated. Another type of
vector is a viral vector, wherein additional DNA segments can be
ligated into the viral genome. Certain vectors are capable of
autonomous replication in a host cell into which they are
introduced (e.g., bacterial vectors having a bacterial origin of
replication and episomal mammalian vectors. Other vectors (e.g.,
non-episomal mammalian vectors) are integrated into the genome of a
host cell upon introduction into the host cell, and thereby are
replicated along with the host genome. Moreover, certain vectors
are capable of directing the expression of genes to which they are
operatively linked. Such vectors are referred to herein as
"expression vectors." In general, expression vectors of utility in
recombinant DNA techniques are often in the form plasmids. In the
present specification, "plasmid" and "vector" can be used
interchangeably as the plasmid is the most commonly used from of
vector. However, the invention is intended to include such other
forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0068] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, which is operatively linked to the nucleic acid
sequence to be expressed. Within a recombinant expression vector,
"operably linked" is intended to mean that the nucleotide sequence
of interest is linked to the regulatory sequence(s) in a manner
which allows for expression of the nucleotide sequence (e.g., in an
in vitro transcription/translation system or in a host cell when
the vector is introduced into the host cell). The term "regulatory
sequence" is intended to include promoters, enhancers, and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel; Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence in many
types of host cell and those which direct expression of the
nucleotide sequence only in certain host cells (e.g.,
tissue-specific regulatory sequences). It will be appreciated by
those skilled in the art that the design of the expression vector
can depend on such factors as the choice of the host cell to be
transformed, the level of expression of protein or RNA desired,
etc. The expression vectors of the invention can be introduced into
host cells to thereby produce siNAs, RNAs, proteins or peptides,
including fusion proteins or peptides.
[0069] In another embodiment, a nucleic acid of the invention is
expressed in mammalian cells using a mammalian expression vector.
The recombinant mammalian expression vector may be capable of
directing expression of the nucleic acid preferentially in a
particular cell type (e.g., tissue-specific regulatory elements are
used to express the nucleic acid). Tissue specific regulatory
elements are known in the art. Non-limiting examples of suitable
tissue-specific promoters include the albumin promoter,
lymphoid-specific promoters, neuron specific promoters, pancreas
specific promoters, and mammary gland specific promoters.
Developmentally-regulated promoters are also encompassed, for
example the murine box promoters and the .quadrature.-fetoprotein
promoter.
[0070] In another aspect the nucleic acid molecules comprise a 5'
and/or a 3'-cap structure. By "cap structure" is meant chemical
modifications, which have been incorporated at either terminus of
the oligonucleotide (see, for example, Wincott et al, WO 97/26270,
incorporated by reference herein). These terminal modifications
protect the nucleic acid molecule from exonuclease degradation, and
can help in delivery and/or localization within a cell. The cap can
be present at the 5'-terminus (5'-cap) or at the 3'-terminus
(3'-cap) or can be present on both terminis. In non-limiting
examples, the 5'-cap includes inverted abasic residue (moiety),
4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide,
4'-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol
nucleotide; L-nucleotides; alpha-nucleotides; modified base
nucleotide; phosphorodithioate linkage; threo-pentofuranosyl
nucleotide; acyclic 3',4'-seco nucleotide; acyclic
3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl
nucleotide, 3'-3'-inverted nucleotide moiety; 3'-3'-inverted abasic
moiety; 3'-2'-inverted nucleotide moiety; 3'-2'-inverted abasic
moiety; 1,4-butanediol phosphate; 3'-phosphoramidate;
hexylphosphate; aminohexyl phosphate; 3'-phosphate;
3'-phosphorothioate; phosphorodithioate; or bridging or
non-bridging methylphosphonate moiety (for more details see Wincott
et al., International PCT Publication No. WO 97/26270, incorporated
by reference herein).
[0071] In another embodiment the 3'-cap includes, for example
4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide;
4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl
phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate;
6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl
phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide;
alpha-nucleotide; modified base nucleotide; phosphorodithioate;
threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide;
3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide,
5'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety;
5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate;
5'-amino; bridging and/or non-bridging 5'-phosphoramidate,
phosphorothioate and/or phosphorodithioate, bridging or
non-bridging methylphosphonate and 5'-mercapto moieties (for more
details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925;
incorporated by reference herein).
[0072] The administration of the herein described nucleic acid
molecules to a patient can be intravenous, intraarterial,
intraperitoneal, intramuscular, subcutaneous, intrapleural,
intrathecal, by perfusion through a regional catheter, or by direct
intralesional injection. When administering these nucleic acid
molecules by injection, the administration may be by continuous
infusion, or by single or multiple boluses. The dosage of the
administered nucleic acid molecule will vary depending upon such
factors as the patient's age, weight, sex, general medical
condition, and previous medical history. Typically, it is desirable
to provide the recipient with a dosage of the molecule which is in
the range of from about 1 pg/kg to 10 mg/kg (amount of agent/body
weight of patient), although a lower or higher dosage may also be
administered.
[0073] A composition is said to be a "pharmaceutically acceptable
carrier" if its administration can be tolerated by a recipient
patient. Sterile phosphate-buffered saline is one example of a
pharmaceutically acceptable carrier. Other suitable carriers are
well-known in the art. See, for example, REMINGTON'S PHARMACEUTICAL
SCIENCES, 18.sup.th Ed. (1990).
[0074] For purposes of immunotherapy, an immunoconjugate and a
pharmaceutically acceptable carrier are administered to a patient
in a therapeutically effective amount. A combination of an
immunoconjugate and a pharmaceutically acceptable carrier is said
to be administered in a "therapeutically effective amount" if the
amount administered is physiologically significant. An agent is
physiologically significant if its presence results in a detectable
change in the physiology of a recipient patient.
[0075] Additional pharmaceutical methods may be employed to control
the duration of action of an immunoconjugate in a therapeutic
application. Control release preparations can be prepared through
the use of polymers to complex or adsorb an immunoconjugate. For
example, biocompatible polymers include matrices of
poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride
copolymer of a stearic acid dimer and sebacic acid. Sherwood et
al., Bio/Technology 10:1446-1449 (1992). The rate of release of
nucleic acid molecules from such a matrix depends upon the
molecular weight of the molecule, the amount of molecule within the
matrix, and the size of dispersed particles. Saltzman et al.,
Biophysical. J. 55:163-171 (1989); and Sherwood et al.,
Bio/Technology 10:1446-1449 (1992). Other solid dosage forms are
described in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed.
(1990).
[0076] Reducing or Eliminating MLCK-L Expression
[0077] The data presented herein establishes the role of the long
MLCK isoform in regulating endothelial permeability in the intact
microcirculation in response to permeability-increasing mediators.
This has been shown using a recently developed strain of mice
lacking the long isoform of MLCK (MLCK2.10.sup.-/- mice) and RNA
silencing to reduce expression of MLCK-L in human endothelial
cells. The findings demonstrate that thrombin-induced increase in
lung microvessel permeability is prevented in MLCK-L.sup.-/- mice.
In addition, using siRNA which specifically knockdown MLCK-L in
human endothelial cells, the results show that MLCK-L suppresses
the increase in intracellular Ca.sup.2+ level induced by thrombin
or thapsigargin by impairing SOC activity. The results identify
MLCK-L as a key effector mediating the increase in vascular
permeability by regulating the phosphorylation of MLC. We uncovered
a novel role of MLCK in the apparently direct activation of SOCs
leading to an MLCK-mediated enhancement of calcium entry.
[0078] Ultrastructural findings in the lung endothelium and
extensive cell culture models have established that cell
contraction leading to gap formation between endothelial cells is a
primary determinant of increased endothelial permeability in
response to agonists such as thrombin, histamine, and VEGF..sup.1
It is known that activation of the PAR-1 receptor by thrombin
within seconds leads to a rise in intracellular Ca.sup.2+ which is
an essential signal inducing activation of the
Ca.sup.2+/CaM-dependent MLCK. MLCK causes actinomyosin stress
fibers to develop force..sup.1 Further, studies showed that
thrombin within minutes also activates the small GTPase RhoA which
induces endothelial contraction. A general and well accepted model
of RhoA regulation of endothelial contraction is that RhoA through
its downstream effector, Rho kinase, can increase MLC
phosphorylation by phosphorylation-mediated inhibition of myosin
light chain phosphatase activity. Thus, intracellular Ca.sup.2+
rise, MLCK and RhoA activation, and endothelial contraction all
precede the formation of interendothelial gaps..sup.1 Studies using
pharmacologic as well as constitutively active recombinant MLCK
protein established that MLCK by phosphorylating MLC is intimately
involved in regulating endothelial permeability in cultured
monolayers or in in vivo models of vascular permeability
regulation..sup.4,5,3,6-10 Likewise, inhibition of both RhoA and
Rho-kinase has also been shown to prevent the increase in
endothelial permeability..sup.1 Thus, the question whether RhoA,
MLCK, or the calcium ion plays the dominant role in controlling MLC
phosphorylation in endothelial cells in response to
permeability-increasing mediators is unresolved. In this regard, we
showed that suppression of endogenous MLCK-L in human endothelial
cells prevented MLC phosphorylation in the presence of normal RhoA
as well as Rho kinase activities. Thus our results support the
concept that MLCK-L activity is required for MLC phosphorylation to
occur and the Rho-ROCK pathway may be able to sustain this
response.
[0079] Studies show that Ca.sup.2+ entry via store-operated cation
(SOC) channels plays a crucial role in sustaining the increase in
intracellular Ca.sup.2+ following depletion of endoplasmic
reticulum (ER) stores..sup.22, 25-30 We found that knock-down of
MLCK-L significantly inhibited SOC induced Ca.sup.2+ entry either
induced by thrombin or thapsigargin. These findings support
previous studies in which pharmacologic inhibition of MLCK was
shown to suppress SOC-mediated Ca.sup.2+ entry in endothelial
cells..sup.31 MLCK-L could regulate the SOC activation either by
phosphorylating the SOC channels or by maintaining their surface
expression..sup.1, 32 Findings from our group as well as others
have implicated the transient receptor potential canonical (TRPC)
channels constitute SOC in endothelial cells..sup.1 Additional
studies will be required to identify the target of MLCK-L.
[0080] Since MLCK-L was required for MLC phosphorylation as well as
SOC activation, we addressed the role of MLCK-L in mediating the
increase in endothelial permeability in cultured cells as well as
intact microcirculation. An important clue that MLCK-L plays an
important role in regulating lung injury was recently provided by
Wainwright et al.sup.6 who developed a strain of mice in which the
long isoform of MLCK was deleted (MLC-LK.sup.-/-). They
demonstrated that in contrast to WT mice i.p. injection of the
bacterial product LPS produced significantly less damage in the
lungs of MLCK-L.sup.-/- mice as reflected by interstitial
hemorrhage, inflammatory cell infiltrate, and atelectasis..sup.6
Furthermore, deletion of MLCK-L in mice enhanced survival during
subsequent mechanical ventilation. Because in addition to
endothelium, MLCK-L may be expressed in other non-muscle cells such
as neutrophils and macrophages, it is possible that the protective
effect of MLCK-L observed in this study could have been due to the
altered function of inflammatory cells. Using an isolated perfused
lung model, we showed that PAR-1 activation did not elicite
increased lung microvessel permeability in MLCK-L.sup.-/- mice. In
addition, we observed that PAR-1-induced MLC phosphorylation was
decreased in lung tissue taken from MLCK-L null mice. We also
showed essentially normal PAR-1-induced pulmonary vasoconstriction
in. MLCK-L deficient mice indicating that a smooth muscle isoform
was functioning in intact lungs as expected. A recent study
similarly found that the MLCK-L deletion did not alter the
contractility response of isolated aortic rings to vasoconstrictor
or vasodilator agonists..sup.33 Thus, the results of the present
study identify the specific role of MLCK-L in the mechanism of
endothelial permeability increase in contrast to the short isoform
specific to vascular smooth muscle cells, which mediates
vasoconstriction. We also demonstrated using TER measurement that
MLCK-L plays an essential role in regulating endothelial barrier
function because knock-down of MLCK-L inhibited TER response in
PAR-1.
[0081] The data herein show that MLCK-L by inducing an increase in
MLC phosphorylation and by augmenting the influx of Ca.sup.2+
through the SOC channel is a key regulator of lung endothelial
permeability.
[0082] Treatment of Endothelial Cells Dysfunctions and Diseases
Thereof Such as Asthma, Adult Respiratory Stress Syndrome, Sepsis
and Trauma
[0083] The vascular endothelium lining the intima of blood vessels
dynamically regulates a variety of cellular functions including
vascular smooth muscle tone, host-defense reactions, wound healing,
angiogenesis, and also provides a semi-selective barrier for tissue
fluid hemostasis. Dysregulation of endothelial function leads to
profound leakage of fluid and macromolecules into the lung tissue
and airspace, resulting in tissue inflammation, a hall mark of
Adult Respiratory Distress Syndrome (ARDS), sepsis and trauma. MLCK
by monophosphorylating the regulatory MLC on Ser19 or by
di-phosphorylating Ser19/Thr18 activates cell contraction which
plays an important role in regulating several endothelial cell
functions such as transendothelial PMN migration elicited by
chemotactic agents and cell survival. Several studies using either
constitutively active MLCK or pharmacological inhibitors of MLCK
have shown that MLCK-induced MLC phosphorylation plays an essential
role in regulating endothelial permeability in vitro and in vivo.
Interestingly, endothelial cells and other inflammatory cells have
been shown to predominantly express the long isoform of MLCK-L.
Small nucleotide profilling of ARDS and asthma patients has
identified MLCK-L to be pathogenic. Results using the mouse model,
as described above, show that siMLCK-L inhibits endotoxin-induced
loss of lung vascular function and, accordingly, indicates
effectiveness of MLCK-L siRNA for clinical applications (treatment)
of disorders and diseases involving endothelial cell dysfunction
such as, but not limited to ARDS, sepsis, trauma, and asthma.
[0084] The invention is further illustrated by way of the following
illustrative examples. However, it is to appreciated that the
invention is not limited to the particular methodologies,
protocols, reagents, results or the like described above and/or set
forth in the illustrative examples. To the contrary, those of skill
in the pertinent arts will readily ascertain from reading the
present disclosure a wide variety of other specific embodiments of
the invention that they can realize without undue experimentation.
A full understanding of the invention and of the claimed subject
matter set forth below may be had only by reading the present
disclosure in full in light of the knowledge and insight of a
person skilled in the arts pertinent thereto.
Examples
Example 1
Materials and Protocols for Assessing Microvessel Permeability in
the Mouse Lung
[0085] Human .alpha.-thrombin was obtained from Enzyme Research
Laboratories (South Bend, Ind.). Human pulmonary arterial
endothelial (HPAE) cells and endothelial growth medium (EBM-2) were
obtained from Clonetics (San Diego, Calif.). Fura 2-AM, BAPTA-AM
and fluorescent antibodies were purchased from Molecular Probes.
Thapsigargin was obtained from Calbiochem (La Jolla, Calif.).
Electrodes for endothelial monolayer electrical resistance
measurements were obtained from Applied Biophysics (Troy, N.Y.).
MLCK-L siRNA were custom synthesized by Dharmacon (Lafayette,
Colo.). Anti-RhoA, anti-VE-cadherin antibodies and transfection
reagents for siRNA were purchased from Santa Cruz Biotechnology
(San Diego, Calif.), phospho-Thr850MYPT1 antibody was purchased
from Upstate (Lake Placid, N.Y.) and anti-MLCK Clone K36 was
purchased from Sigma (St Louis, Mo.). Rho activity was determined
using GST-rhotekin-Rho-binding domain beads from Cytoskeleton
(Denver, Colo.). Anti-phosho-MLC antibody was a gift from Dr.
Jerold Turner (University of Chicago).
[0086] Endothelial Cell Culture
[0087] HPAE cells were cultured in T-75 flasks coated with 0.1%
gelatin in EBM-2 medium supplemented with 10% FBS. In all
experiments, HPAE cells between passage 6 and 8 were used.
[0088] Microvessel Permeability Studies in Mouse Lung
[0089] We measured microvessel permeability in the wild type and
MLCK210.sup.-/- mice lung by determining microvascular filtration
coefficient (k.sub.f,c) as described..sup.20 Briefly, according to
an approved protocol of the University of Illinois at Chicago
Animal Care Committee, male mice weighing 25-30 g were anesthetized
with a combination of 10 mg/ml ketamine, 0.25 mg/ml xylazine, and
0.25 mg/ml acepromazine and isolated mouse lungs were prepared and
perfused as described..sup.20 All lung preparations underwent a 20
min equilibration perfusion, and lungs that were not isogravimetric
at the end of this period were discarded. After a 20-min
equilibration perfusion, outflow pressure was elevated by 10 cm
H.sub.2O for 2 min. The lung wet weight increase over this time,
which reflects the net fluid accumulation, was continuously
recorded. At the end of each experiment, lung dry weight was
determined. K.sub.f,c (mlmin.sup.-1cmH.sub.2Og dry wt.sup.-1) was
calculated from the slope of the recorded weight change normalized
to the pressure change and lung dry weight. The expression of
MLCL-L in lungs was determined by Western blotting of lung
homogenate with anti-MLCK Ab.
[0090] Transfection of siRNA
[0091] siRNAs were transfected into cells using Santa Cruz
transfection reagent following manufacture's protocol.
[0092] Western Blotting
[0093] Lysates from HPAE cell monolayers were Western blotted with
indicated antibodies using published protocols..sup.21
[0094] Cytosolic Ca.sup.2+ Measurements
[0095] An increase in intracellular Ca.sup.2+ was measured using
the Ca.sup.2+-sensitive fluorescent dye Fura 2-AM as
described..sup.21, 22
[0096] Measurements of RhoA Activity
[0097] RhoA activity was measured using GST-rhotekin-Rho binding
domain (GST-RBD) that specifically pulls down activated RhoA as
described..sup.21, 22
[0098] Immunoflorescence
[0099] After stimulation with thrombin, cells were rinsed quickly
with ice-cold HBSS, fixed and stained with anti-VE-cadherin Ab
followed by alexa-labeled secondary Ab as described
previously..sup.21
[0100] MLC Phosphorylation
[0101] Cells stimulated with thrombin were lysed with Laemmli
sample buffer and western blotted with antibodies for
phosphorylated-MLC or pan-MLC Abs to determine MLC
phosphorylation.
[0102] Transendothelial Electrical Resistance Measurement
[0103] The time course of endothelial cell retraction, a measure of
increased endothelial permeability, was measured using established
protocols..sup.23
[0104] Statistical Analysis
[0105] Two-tailed Student t-test and one-way ANOVA with Bonferroni
post-hoc test were used for statistical comparisons. Differences
were considered significant at P<0.05.
Example 2
MLCK-L Knockdown Prevents MLC Phosphorylation Without Altering RhoA
Activation
[0106] Three siRNA sequences corresponding to the N-terminal region
of MLCK-L were constructed. Western blot analysis showed that siRNA
Seq 3 reduced MLCK-L expression by .about.75% within 48 hr (FIG.
1A-B). Seq 1, by contrast, was ineffective (FIG. 1A-B). Thus, in
the remainder of the study, we used Seq 1 as control for comparison
with Seq 3 (i.e. MLCK-L siRNA) to address the role of MLCK-L in
regulating endothelial barrier function. Thrombin induced MLC
phosphorylation in cells transfected with control siRNA (FIG. 2).
However, "knockdown" of MLCK-L prevented MLC phosphorylation (FIG.
2A-B). A recent study showed that Rho kinase can also phosphorylate
MLC..sup.24 To see if the observed inhibition of MLC
phosphorylation is due to altered RhoA-Rho kinase activity in
MLCK-L knock-down cells, we performed a Rho activity assay and also
determined phosphorylation of MYTP-1, a specific Rho kinase
substrate. Thrombin induced equivalent RhoA activation in control
and in MLCK-L knockdown cells (FIG. 3A). We determined the extent
of MYPT1 phosphorylation using an anti-phospho-thr850 MYPT1
antibody. Suppression of MLCK-L had no effect on thrombin-induced
MYPT1 phosphorylation (FIG. 3B-C). Thus, these results indicate the
essential role of MLCK-L in inducing MLC phosphorylation
independently of the Rho/Rho kinase pathway.
Example 3
Impaired Ca2+ Influx in MLCK-L Knock-Down Endothelial Cells in
Response to Thrombin or Thapsigargin
[0107] We investigated whether calcium entry calcium entry through
store-operated channels (required for endothelial monolayer
retraction) is impaired in MLCK-deficient endothelial cells. We
therefore determined whether suppression of endogenous MLCK-L
affects store-operated calcium entry channels (SOCs) in Fura-2
loaded endothelial monolayer. We used thrombin to deplete the ER
calcium store and elicit calcium entry. We separately assessed the
calcium-release and calcium-entry components using a Ca.sup.2+
add-back protocol, which allowed us to determine the role of MLCK-L
in regulating SOC activation. We observed that under Ca.sup.2+-free
bath conditions, MLCK-L knockdown had no effect on thrombin-induced
release of Ca.sup.2+-from stores (FIG. 4A-B). However, suppression
of MLCK-L expression significantly reduced Ca.sup.2+ entry upon
bath-calcium repletion (FIG. 4A-B).
[0108] We used thapsigargin for comparison with thrombin, since
thapsigargin activates SOCs independently of ligand-receptor-G
protein-coupled receptors. Thapsigargin increased Ca.sup.2+ entry
via SOCs (FIG. 4C-D), as expected. However, suppression of MLCK-L
inhibited SOC activation (FIG. 4C-D). These findings thereby
indicate that MLCK-L regulates SOC channels independently of
G-protein signaling downstream of the PAR-1 receptor.
Example 4
MLCK-L Knockdown Prevents Cell Retraction in Response to
Thrombin
[0109] Since our studies show that MLCK-L deficiency led to
suppression of thrombin-induced calcium entry and MLC
phosphorylation, we next measured endothelial cell retraction and
paracellular permeability increase in response to thrombin. We
determined adherens junction organization to assess the effect of
MLCK-L knock-down on thrombin-induced junctional disassembly, a
prerequisite for the increase in endothelial permeability. We also
determined transendothelial electrical resistance (TER) in
endothelial monolayers to address the role of MLCK-L in regulating
endothelial permeability. As shown in FIG. 5, thrombin induced the
disruption of adherens junction in cells transfected with control
siRNA. However, this response was not seen in cells transfected
with MLCK-L siRNA. In endothelial monolayers transfected with
control siRNA, thrombin caused a decrease in transendothelial
monolayer electrical resistance of .apprxeq.55% (FIG. 6). By
contrast, exposure of thrombin to MLCK-L knockdown cells produced
only a .apprxeq.15% decrease in resistance after thrombin challenge
(FIG. 6). Thus, these findings identify the specific role of MLCK-L
as a key regulator of endothelial barrier function in response to
thrombin.
Example 5
Deletion of MLCK-L in Mice Prevents Increase in Lung Vascular
Permeability in Response to Selective PAR-1 Receptor Agonist
TFLLRNPNDK (SEQ ID NO: 5)
[0110] We measured microvessel liquid permeability in isolated lung
preparations from wild-type (WT) and MLCK-L.sup.-/- mice to
determine the role of MLCK-L in regulating lung vascular
permeability (FIG. 7A, inset). We found that basal values of
K.sub.f,c did not significantly differ between WT and
MLCK-L.sup.-/- lungs (FIG. 7A). In wt mice, PAR-1 activation
produced a 2-3-fold increase in lung K.sub.f, c within 15 minutes
(FIG. 7A). However, this response was not observed in lungs from
MLCK-L.sup.-/- mice. We also determined PAR-1-induced pulmonary
vasoconstriction in the MLCK-L.sup.-/- mice. Thrombin-induced
vasoconstriction was the same in MLCK-L.sup.-/- mouse lung as in
the WT mice lung. Thus, these results identify the specific role of
MLCK-L.sup.-/- in the mechanism of endothelial permeability
increase in contrast to the other isoform specific to vascular
smooth muscle cells, which mediates vasoconstriction (FIG. 7B).
[0111] We next determined the effect of thrombin on the extent of
MLC phosphorylation in mouse lung tissue pretreated with PAR-1
agonist to address the possibility that the impairment in lung
vascular permeability is the result of inhibition of MLC
phosphorylation. Western immunoblots show MLC and phosphorylated
MLC (FIG. 8). In lung tissue obtained from normal mice, thrombin
induced an increase in MLC phosphorylation. Induction of MLC
phosphorylation by thrombin was decreased in lung tissue taken from
MLCK-L null mice, indicating the essential role of MLC
phosphorylation induced by MLCK-L in mediating the thrombin
action.
Example 6
Reduction in Eye Inflammation
[0112] We observed a significant inhibition of the eye inflammation
in the mouse lacking the MLCK gene. Wild type or MLCK-L knockout
mice were anesthetized following protocol approved by UIC Animal
Care Committee. The subconjunctival scarring was generated by
injecting 30 .mu.l of PBS containing latex beads in the temporal
subconjunctival space of the mouse's left eye. The beads were 1.053
.mu.m in diameter, 300 .mu.g/ml (Polyscience, Warrington, Pa.). The
right eye, which received no injection, served as an internal
control in each group. One-week post treatment mice were sacrificed
by cervical dislocation. Eyes were removed by enucleation and fixed
in 10% buffered folinalin and 5 .mu.m thick paraffin sections were
prepared. The sections were stained with hematoxylin and eosin to
assess the inflammatory reaction and picrocirius red to visualize
the collagen deposit. The numbers of inflammatory cells in
subconjunctival areas in the sections were counted. The value was
normalized to the number of inflammatory cells per unit area (2500
.mu.m.sup.2) underneath the conjunctival epithelium. For each
treatment at least 4 mice were used. See FIG. 9.
Example 10
Suppression of Endogenous MLCK-L in Mice Events LPS-Induced Lung
Injury
[0113] We used MLCK siRNA to address the possibility that knockdown
of MLCK-L protects mouse against LPS, a bacterial endotoxin,
induced lung injury. As shown in FIG. 1, transduction of MLCK-L
siRNA (but not control siRNA) decreased MLCK-L (210) but not smooth
muscle isoform (130) expression after 48 hr of transfection. We
observed that in mice injected with control siRNA, administration
of LPS induced a marked increase in lung inflammation as indicated
by changes in the lung wet- to dry weight ratio, albumin
accumulation in the lung and increased number of inflammatory cells
in the bronchoalveolar lavage of mice. However, LPS-induced lung
injury was significantly reduced in mice injected with MLCK-L
siRNA. See FIG. 10.
REFERENCES
[0114] Publications (references) cited herein and the material for
which they are cited are specifically incorporated by reference.
Nothing herein is to be construed as an admission that the
publication is prior art to the invention herein disclosed and
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Sequence CWU 1
1
4121RNAArtificialMLCK-L siRNA 1nnggacugcg cuguuauuga g
21221RNAArtificialMLCK-L siRNA 2nnguggaaag gcuugccgug a
21321RNAArtificialMLCK-L siRNA 3nnugggcagc ccauccagua c
2144740DNAHomo sapiens 4ccatggggga tgtgaagctg gttgcctcgt cacacatttc
caaaacctcc ctcagtgtgg 60atccctcaag agttgactcc atgcccctga cagaggcccc
tgctttcatt ttgccccctc 120ggaacctctg catcaaagaa ggagccaccg
ccaagttcga agggcgggtc cggggttacc 180cagagcccca ggtgacatgg
cacagaaacg ggcaacccat caccagcggg ggccgcttcc 240tgctggattg
cggcatccgg gggactttca gccttgtgat tcatgctgtc catgaggagg
300acaggggaaa gtatacctgt gaagccacca atggcagtgg tgctcgccag
gtgacagtgg 360agttgacagt agaaggaagt tttgcgaagc agcttggtca
gcctgttgtt tccaaaacct 420taggggatag attttcagct tcagcagtgg
agacccgtcc tagcatctgg ggggagtgcc 480caccaaagtt tgctaccaag
ctgggccgag ttgtggtcaa agaaggacag atgggacgat 540tctcctgcaa
gatcactggc cggccccaac cgcaggtcac ctggctcaag ggaaatgttc
600cactgcagcc gagtgcccgt gtgtctgtgt ctgagaagaa cggcatgcag
gttctggaaa 660tccatggagt caaccaagat gacgtgggag tgtacacgtg
cctggtggtg aacgggtcgg 720ggaaggcctc gatgtcagct gaactttcca
tccaaggttt ggacagtgcc aataggtcat 780ttgtgagaga aacaaaagcc
accaattcag atgtcaggaa agaggtgacc aatgtaatct 840caaaggagtc
gaagctggac agtctggagg ctgcagccaa aagcaagaac tgctccagcc
900cccagagagg tggctcccca ccctgggctg caaacagcca gcctcagccc
ccaagggagt 960ccaagctgga gtcatgcaag gactcgccca gaacggcccc
gcagaccccg gtccttcaga 1020agacttccag ctccatcacc ctgcaggccg
caagagttca gccggaacca agagcaccag 1080gcctgggggt cctatcacct
tctggagaag agaggaagag gccagctcct ccccgtccag 1140ccaccttccc
caccaggcag cctggcctgg ggagccaaga tgttgtgagc aaggctgcta
1200acaggagaat ccccatggag ggccagaggg attcagcatt ccccaaattt
gagagcaagc 1260cccaaagcca ggaggtcaag gaaaatcaaa ctgtcaagtt
cagatgtgaa gtttccggga 1320ttccaaagcc tgaagtggcc tggttcctgg
aaggcacccc cgtgaggaga caggaaggca 1380gcattgaggt ttatgaaaat
gctggctccc attacctctg cctgctgaaa gcccggacca 1440gggacagtgg
gacatacagc tgcactgctt ccaacgccca aggccaggtg tcctgtagct
1500ggaccctcca agtggaaagg cttgccgtga tggaggtggc cccctccttc
tccagtgtcc 1560tgaaggactg cgctgttatt gagggccagg attttgtgct
gcagtgctcc gtacggggga 1620ccccagtgcc ccggatcact tggctgctga
atgggcagcc catccagtac gctcgctcca 1680cctgcgaggc cggcgtggct
gagctccaca tccaggatgc cctgccggag gaccatggca 1740cctacacctg
cctagctgag aatgccttgg ggcaggtgtc ctgcagcgcc tgggtcaccg
1800tccatgaaaa gaagagtagc aggaagagtg agtaccttct gcctgtggct
cccagcaagc 1860ccactgcacc catcttcctg cagggcctct ctgatctcaa
agtcatggat ggaagccagg 1920tcactatgac tgtccaagtg tcagggaatc
caccccctga agtcatctgg ctgcacaatg 1980ggaatgagat ccaagagtca
gaggacttcc actttgaaca gagaggaact cagcacagcc 2040tttgtatcca
ggaagtgttc ccggaggaca cgggcacgta cacctgcgag gcctggaaca
2100gcgctggaga ggtccgcacc caggccgtgc tcacggtaca agagcctcac
gatggcaccc 2160agccctggtt catcagtaag cctcgctcag tgacagcctc
cctgggccag agtgtcctca 2220tctcctgcgc catagctggt gacccctttc
ctaccgtgca ctggctcaga gatggcaaag 2280ccctctgcaa agacactggc
cacttcgagg tgcttcagaa tgaggacgtg ttcaccctgg 2340ttctaaagaa
ggtgcagccc tggcatgccg gccagtatga gatcctgctc aagaaccggg
2400ttggcgaatg cagttgccag gtgtcactga tgctacagaa cagctctgcc
agagcccttc 2460cacgggggag ggagcctgcc agctgcgagg acctctgtgg
tggaggagtt ggtgctgatg 2520gtggtggtag tgaccgctat gggtccctga
ggcctggctg gccagcaaga gggcagggtt 2580ggctagagga ggaagacggc
gaggacgtgc gaggggtgct gaagaggcgc gtggagacga 2640ggcagcacac
tgaggaggcg atccgccagc aggaggtgga gcagctggac ttccgagacc
2700tcctggggaa gaaggtgagt acaaagaccc tatcggaaga cgacctgaag
gagatcccag 2760ccgagcagat ggatttccgt gccaacctgc aacggcaagt
gaagccaaag actgtgtctg 2820aggaagagag gaaggtgcac agcccccagc
aggtcgattt tcgctctgtc ctggccaaga 2880aggggacttc caagaccccc
gtgcctgaga aggtgccacc gccaaaacct gccaccccgg 2940attttcgctc
agtgctgggt ggcaagaaga aattaccagc agagaatggc agcagcagtg
3000ccgagaccct gaatgccaag gcagtggaga gttccaagcc cctgagcaat
gcacagcctt 3060cagggccctt gaaacccgtg ggcaacgcca agcctgctga
gaccctgaag ccaatgggca 3120acgccaagcc tgccgagacc ctggagccca
tgggcaatgc caagcctgat gagaaccaat 3180aatccgctag caaagaagaa
ctcaagaaag acgttaagaa tgatgtgaac tgcaagagag 3240gccatgcagg
gaccacagat aatgaaaaga gatcagagag ccaggggaca gccccagcct
3300tcaagcagaa gctgcaagat gttcatgtgg cagagggcaa gaagctgctg
ctccagtgcc 3360aggtgtcttc tgacccccca gccaccatca tctggacgct
gaacggaaag accctcaaga 3420ccaccaagtt catcatcctc tcccaggaag
gctcactctg ctccgtctcc atcgagaagg 3480cactgcctga ggacagaggc
ttatacaagt gtgtagccaa gaatgacgct ggccaggcgg 3540agtgctcctg
ccaagtcact gtggatgatg ctccagccag tgagaacacc aaggccccag
3600agatgaaatc ccggaggccc aagagctctc ttcctcccgt gctaggaact
gagagtgatg 3660cgactgtgaa aaagaaacct gcccccaaga cacctccgaa
ggcagcaatg ccccctcaga 3720tcatccagtt ccctgaggac cagaaggtac
gcgcaggaga gtcagtggag ctgtttggca 3780aagtgacagg cactcagccc
atcacctgta cctggatgaa gttccgaaag cagatccagg 3840aaagcgagca
catgaaggtg gagaacagcg agaatggcag caagctcacc atcctggccg
3900cgcgccagga gcactgcggc tgctacacac tgctggtgga gaacaagctg
ggcagcaggc 3960aggcccaggt caacctcact gtcgtggata agccagaccc
cccagctggc acaccttgtg 4020cctctgacat tcggagctcc tcactgaccc
tgtcctggta tggctcctca tatgatgggg 4080gcagtgctgt acagtcctac
agcatcgaga tctgggactc agccaacaag acgtggaagg 4140aactagccac
atgccgcagc acctctttca acgtccagga cctgctgcct gaccacgaat
4200ataagttccg tgtacgtgca atcaacgtgt atggaaccag tgagccaagc
caggagtctg 4260aactcacaac ggtaggagag aaacctgaag agccgaagga
tgaagtggag gtgtcagatg 4320atgatgagaa ggagcccgag gttgattacc
ggacagtgac aatcaatact gaacaaaaag 4380tatctgactt ctacgacatt
gaggagagat taggatctgg gaaatttgga caggtctttc 4440gacttgtaga
aaagaaaact cgaaaagtct gggcagggaa gttcttcaag gcatattcag
4500caaaagagaa agagaatatc cggcaggaga ttagcatcat gaactgcctc
caccccccta 4560agctggtcca gtgtgtggat gcctttgaag aaaaggccaa
catcgtcatg gtcctggaga 4620tcgtgtcagg aggggagctg tttgagcgca
tcattgacga ggactttgag ctgacggagc 4680gtgagtgcat caagtacatg
cggcagatct cggagggagt ggagtacatc cacaagcagg 4740
* * * * *